Introduction
Oxidative stability is an important parameter in determining quantitatively the deterioration rate of oils/fats (Laubli et al., "Determination of the Oxidative Stability of Fats and Oils: Comparison Between the Active Oxygen Method (AOCS Cd-12-57) and the Rancimat Method," J. Am. Oil Chem. Soc., 63:792-795 (1986) ("Laubli"), which is hereby incorporated by reference). This measurement is crucial to both oil processors and users in specifying and examining the quality of their products. Deep-fat frying is one of the major oil applications in the United States (Stevenson et al., "Quality Control in the Use of Deep Frying Oil," J. Am. Oil Chem. Soc., 61:1102-1108 (1984) ("Stevenson"), which is hereby incorporated by reference). In 1992, 38.4% of the total amount (2.4 million kg) of edible fats/oils consumed by Americans was used for frying and baking (USDA, "Oil Crops Situations and Outlook," October 1992. OCS-35. Dept. of Agriculture, Washington, D.C., which is hereby incorporated by reference). The edible oil industry has long sought for a rapid method to predict oxidative stability of fats/oils (Hill, "Comparisons: Measuring Oxidative Stability," Inform, 5:104-109 (1994) ("Hill"), which is hereby incorporated by reference); however, the most up-to-date instrument, the Oil Stability Instrument, requires many hours (10 to over 100) to complete an analysis. A rapid and accurate method for such a measurement is needed to provide both time efficiency and accurate results for the oil industry.
Oxidative decomposition of a fat/oil is detrimental to the acceptability and nutritional quality of foods (Nawar, "Lipid," Ch. 4. in Food Chemistry, 2.sup.nd ed. O. R. Fennema (ed.), New York: Marcel Dekker, nc. ("Nawar"), which is hereby incorporated by reference). The major causes of the decomposition of a fat/oil are lipid hydrolysis and autoxidation (Arroyo et al., "High-Performance Size-Exclusion Chromatographic Studies on Polar components Formed in Sunflower Oil Used for Frying," J. Am. Oil Chem. Soc., 69:557-563 (1992) ("Arroyo"); Frankel, "Recent Advances in Lipid Oxidation," J. Sci. Food Agric., 54:495-511 (1991), which are hereby incorporated by reference). Many methods have been designed to measure the stability of a fat/oil based on the chemical or physical changes caused by the decomposition of the fat/oil. These methods such as the Active Oxygen Method ("AOM"), peroxide value ("PV"), thiobarbituric acid ("TBA") value, iodine value ("IV"), Shaal oven test, oxygen adsorption, chromatographic methods, and thermogravimetric analysis generally are used for evaluating the oxidative stability of a fat/oil (deMan et al., "Formation of short Chain Volatile Organic Acids in the Automated AOM Method," J. Am. Oil Chem. Soc., 64:993-996 (1987) ("deMan"); Mikula et al., "Reaction Conditions for Measuring Oxidative Stability of Oils by Thermogravimetric Analysis," J. Am. Oil Chem. Soc., 62:1694-1698 (1985) ("Mikula"), which is hereby incorporated by reference; Nawar). However, none of these tests are fully representative of oxidative stability of a fat/oil in terms of practical applications such as frying and cooking (Nawar). These tests are time consuming, labor intensive, and costly (Laubli). Therefor, a low-cost, rapid, accurate, and precise instrument for fat/oil oxidative stability measurement is needed.
AOM is the most popular method for determining the oxidative stability of a fat/oil (Hill). the oil sample (5 g) is heated to 98.7.degree. C. and bubbled with a stream of dry air (140 mL/min flow rate). AOM value is determined by the time at which the oil sample reaches 100 meq of peroxide per kg of sample (AOCS Cd 12-57; AOCS, Official and Tentative Methods of Analysis of the American Oil Chemists Society, American Oil Chemists Society, Champaign, Ill. 1993), which is hereby incorporated by reference). The Rancimat method and Oil Stability Instrument are modified versions of AOM but use a higher temperature than that used by AOM. These instruments measure the increases of conductivity in water, caused by oxidation products such as volatile acids, to determine the oxidative stability (Hill; Laubli). AOM and its modified versions are either costly or time consuming.
Drozdowski et al., "A Rapid Instrumental Method for the Evaluation of the Stability of Fats," J. Am. Oil Chem. Soc., 64:1008-1010 (1987) ("Drozdowski"), which is hereby incorporated by reference, analyzed the resistance to oxidation of oils by an oxygen adsorption instrument. They reported large variations in amounts of oxygen adsorption among 4 oil samples that had similar fatty acid composition, iodine value, and peroxide value. This method is rapid but expensive, and the accuracy of the results is questionable. Snyder et al., "Headspace Volatile Analysis to Evaluate Oxidative and Thermal Stability of Soybean Oil. Effect of Hydrogenation and Additives," J. Am. Oil Chem. Soc., 62:1675-1679 (1986) ("Snyder"), which is hereby incorporated by reference, used gas chromatography ("GC") to measure the volatiles in the headspace of heated oil samples. They reported that the concentrations of volatiles formed in the headspace had good correlations with oil stability. However, their method needs sophisticated instrumentation and cannot handle a large volume of samples. Garcia-Mesa et al., "Factors Affecting the Gravimetric Determination of the Oxidative Stability of Oils," J. Am. Oil Chem. Soc., 70:245-247 (1993) ("Garcia-Mesa"), which is hereby incorporated by reference, used a gravimetric method to determine the oxidative stability of oil; the weight gains by oil samples heated in the hot air oven were positively correlated (P&lt;0.05) with peroxide values. This method requires intensive work; the samples must be weighed frequently and each test requires at least 20 hr.
In addition to the disadvantages of low time efficiency and high cost, the methods mentioned above overlook the hydrolysis occurring in fats/oils in a frying system. An ideal method for evaluating the stability of a fat/oil under frying conditions should look into all chemical reactions occurring during that process. For example, the frying oil is exposed to air, moisture, and high temperatures (160-190.degree. C.). The moisture causes hydrolysis of triglycerides, producing free fatty acids and mono- and diglycerides. The air, absorbed by the oil, initiates oxidative decomposition. High frying temperatures (160-190.degree. C.) can trigger the thermal decomposition in the fats/oils (Arroyo; Boskou, "Stability of Frying Oils," Varela, eds., Frying of Food: Principles, Changes, New Approaches, Chichester, England: Ellis Horwood Ltd., ch. 13, pp. 174-182 (1988) ("Boskou"), which is hereby incorporated by reference). Frying fats come into contact with moisture, which is released from food during frying. While this moisture can increase in free fatty acids in the frying oils, it also aids in removing other oxidative degradation products from the oil via steam distillation. This stem distillation effect actually prolongs the useful life of a frying fat. All these reactions occurring in fats/oils during frying should be taken into consideration when building a reliable instrument for measuring oil oxidative stability.
A prototype instrument 20 for measuring frying oil stability made in accordance with the present invention was developed in The University of Tennessee Food Science and Technology department. The instrument 20 permits oxidation of fats at frying temperatures while moisturized air is bubbled through the hot oil. This computer-based instrument 20 is similar to the Oil Stability Instrument in that it measures the increase in conductivity of water-trapped decomposition products from the oil, but it has several unique differences, which are further discussed below.
Literature Review
1. Chemical changes in the Lipid During Frying
Lipid oxidation is preceded primarily by an autoxidation mechanism and initiated by free radicals. The production of free radicals may occur by thermolysis (thermal dissociation), hydroperoxide decomposition, metal catalysis, and exposure to light (photolysis) with or without initiation by photosensitizers. Autoxidation undergoes a chain mechanism of three stages and includes initiation, propagation, and termination. Hydroperoxides are primary products of lipid oxidation. Due to their unstable nature, hydroperoxides readily break down and produce free radicals, alcohols, aldehydes, and ketones; these decomposition compounds can undergo further oxidation to produce carboxylic acids or they may polymerize (Frankel, "Lipid Oxidation," Lipid Res., 19:1-22 (1980), which is hereby incorporated by reference; Laubli; Lin, "Flavor and Stability of Potato Chips Fried in Canola, High Oleic Sunflower, Sunflower, and Cottenseed Oils," Master's Thesis, the University of Tennessee, Knoxville (1983) ("Lin"), which is hereby incorporated by reference).
A variety of chemical reactions occur in the oil/fat during the frying process in which the fat/oil is subjected to air, high temperature, and steam. The air incorporated into the frying media is an oxygen source for oxidation and triggers the formation of free radicals. Steam causes hydrolysis of triglycerides (Cuesta et al., "Thermoxidative and Hydrolytic Changes in Sunflower Oil Used in Frying With a Fast Turnover of Fresh Oil," J. Am. Oil Chem. Soc., 70:1069-1073 (1993) ("Cuesta"), which is hereby incorporated by reference). A high frying temperature provides energy to favor chemical reactions and causes thermolysis. In general, unsaturated fatty acids in oil/fat undergo the autoxidation reaction by a free radical mechanism. However, even saturated fats may undergo thermal decomposition at frying temperatures (Frank et al., "Automatic Determination of Oxidation Stability of Oil and Fatty Products," Food Technol., 35:71-76 (1982), which is hereby incorporated by reference; Lin; Paquette et al., "The Mechanisms of Lipid Autoxidation. I. Primary Oxidation Products," Can. Inst. Food Sci. Technol., 18:112-118 (1985), which is hereby incorporated by reference). In addition to the decomposition compounds formed during lipid oxidation, hydrolysis of triglycerides also causes formation of free fatty acids, glycerol, and mono- and di-glycerides (Cuesta).
In general, decomposition compounds of oil/fat oxidation are categorized into volatile decomposition products ("VDP") and non-volatile decomposition products ("NVDP"). These compounds usually are used as the indicators of oil/fat deterioration. Many factors, such as type of frying fat, type of food, conditions of operation, and type of fryer can cause variations in the physical and chemical changes occurring in frying fat during deep-fat frying (Fritsch, "Measurement of Frying Fat Deterioration: A Brief Review," J. Am. Oil Chem. Soc., 58:272-274 (1981) ("Fritsch"), which is hereby incorporated by reference).
Cuesta used refined sunflower oil to fry potatoes continuously with rapid turnover rate. They discovered more thermoxidative than hydrolytic reactions during deep-fat frying. Total polar components ("TPC") level was used as a representative measurement of the total alterations of the oil. Oxidized, dimeric, and polymeric triglycerides indicated thermoxidative alteration of the oil. Free fatty acids plus diglycerides represented the major products of hydrolytic reaction. The relationship between TPC levels and total thermoxidative alteration in the frying oil or numbers of times used for frying could be explained by third order polynomial regression equations, in which the regression coefficients were 0.9957 and 0.9949, respectively. TPC level and polymer content increased rapidly during the earlier frying stage then tended to level off during the latter stage of frying. The diglyceride content and total hydrolytic modifications (diglycerides plus free fatty acids) in the frying oil increased slightly as the number of times the oil was used for frying increased, but the magnitudes of the increases with increasing number of times that the oil was used for frying were not statistically significant. The relationship between free fatty acid content and the number of frying times was irrelevant because free fatty acids were partially lost to the atmosphere during frying. Other researchers also noted that the linoleic acid content of the frying oil decreased while oleic, stearic, and palmitic acids remained unaltered after the sunflower oil was used for frying potatoes for 15 times (Arroyo; Cuesta; Sanchez-Muniz et al., "Sunflower Oil Used for Frying: Combination of Column, Gas and High-Performance Size-Exclusion Chromatography for its Evaluation," J. Am. Oil Chem. Soc., 70:235-240 (1993) ("Sanchez-Muniz"), which is hereby incorporated by reference).
Farag et al, "Comparative Study on the Deterioration of Oils by Microwave and Conventional Heating," J. Food Prot., 55:722-727 (1992), which is hereby incorporated by reference, heated oil by microwave and deep-fat frying. They discovered a significant decrease in levels of oleic and linoleic acids and an increase in the amount of palmitic acid in heated refined cottonseed oil. They explained that the heating process may have caused an abstraction of a hydrogen atom from the active methylene group adjacent to the carboxyl group to produce free radicals, followed by oxidative degradation to produce shorter chain fatty acids such as palmitic and acetic acids.
Smith et al., "changes in Physical and Chemical Properties of Shortenings Used for Commercial Deep-Fat Frying," J. Am. Oil Chem. Soc. 63:1017-1023 (1986) ("Smith"), which is hereby incorporated by reference, surveyed the quality of the oils used for commercial deep-fat frying. A variety of oil quality indicators such as free fatty acids ("FFA"), TPC, fatty acid profile, and dielectric constant (measured as the Food Oil Sensor, or FOS, reading) were determined and compared with the number of frying times of the samples. They observed marked increases of dielectric measurements, TPC, and FFA in the used oil samples. The greatest change in fatty acid profiles occurred in the trans-C18:1 fatty acid level, which decreased over 40%. Stearic acid (C18:0) concentration also decreased during frying while level of C16:0 increased. Increase of C18:1 and C18:2 over frying times was not due to the effects of frying but to an exchange of the frying fat with the lipid in the foods.
2. Effects of Antioxidant on Retardation of Lipid Oxidation
Antioxidants retard the deterioration of lipid oxidation by interfering with free radical formation or quenching a pro-oxidant such as singlet oxygen. Antioxidants such as butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), butylated hydroquinone ("TBHQ"), and propyl gallate interfere with free radicals by a chain-breaking mechanism during initiation or propagation stage of lipid oxidation. The antioxidant compounds mentioned above generally lose their efficiency at elevated temperatures by homolytic decomposition of hydroperoxides or by reaction with oxygen (Frankel, "Lipid Oxidation," Lipid Res., 19:1-22 (1980), which is hereby incorporated by reference). Tocopherols have been reported as free radical scavengers. The (.alpha.-tocopherol is known for quenching singlet oxygen, considered as a pro-oxidant formed by photochemical sensitizers such as chlorophylls. The amounts of .alpha.-, .beta.-, .gamma.-, and .delta.-tocopherols present in soybean oil are reported as 91-118, 21-43, 640-795, and 325-406 ppm, respectively. The optimum concentration of total tocopherols for retarding lipid oxidation in a soybean oil is between 400 and 600 ppm (Jung et al., "Effects of .alpha.-, .gamma.-, and .delta.-Tocopherol on Oxidative Stability of Soybean Oil," J. Food Sci., 55:1464-1465 (1990) ("Jung I"); Jung et al., ".alpha.-, .gamma.-, and .delta.-Tocopherol Effects on Chlorophyll Photosensitized Oxidation of Soybean Oil," J. Am. Oil Chem. Soc., 56:807-810, 815 (1991) ("Jung II"); Weiss, Foods Oils and Their Uses, 2.sup.nd Ed., Westport, Conn.: AVI Publishing Co., Inc. (1983) ("Weiss"), which are hereby incorporated by reference).
Asap et al., "Effect on TBHQ on Quality Characteristics of RBD Olein During Frying," J. Am. Oil Chem. Soc., 63:1169-1175 (1986), which is hereby incorporated by referenced, examined the effect of TBHQ on the quality characteristics, including TPC, iodine value ("IV"), and fatty acid profile, of refined, bleached, and deodorized ("RBID") palm olein during frying. Treatments included an initial 200 ppm of TBHQ with or without replenishment of the loss of TBHQ during each day of frying. Results were compared to those of a control sample. The authors concluded that the addition of TBHQ reduced the levels of TPC and polymers in the oil, decreased the rate of change in iodine value ("IV") and dielectric constant, and decreased the rate of C18:2 oxidation. When the loss of TBHQ during frying was replenished each day, the reduction rates of the undesirable changes were even more profound than without the replenishment of TBHQ loss.
Augustin et al., "Efficacy of the Antioxidants BHA and BHT in Palm Oleic During Heating and Frying," J. Am. Oil Chem. Soc., 60:1520-1523 (1983), which is hereby incorporated by referenced, assessed the effectiveness of BHA and BHT in retarding the deterioration of RBD palm olein during static heating (180.degree. C.) and frying of potato chips; the effectiveness was assessed by peroxide value ("PV"), anisidine value, FFA level, IV, fatty acid profile, and dienoic acid level. In general, PV, anisidine value, FFA, and dienoic acids increased over the period of heating or frying while the IV and ratio of C18:2/C16:0 decreased. Heated oil had lower rates of the changes of the above measurements than did the oil used for frying. The authors noted also that BHA was more effective than BHT in retarding oil oxidation during static heating, whereas, both BHA and BHT became ineffective for antioxidative function during intermittent frying of potato chips.
Jung I examined the effectiveness of .alpha.-, .gamma.-, and .delta.-tocopherons at various concentrations on oxidative stability of soybean oil stored in the dark at 55.degree. C. PV and headspace oxygen consumption were used to measure the effectiveness. Tocopherols had antioxidative effects at the lower concentrations but acted as pro-oxidants at higher concentrations. The optimum concentrations of (.alpha.-, .gamma.-, and .delta.-tocopherol for antioxidative effect on purified soybean oil were 100, 250, and 500 ppm, respectively. In other words, under these conditions, .alpha.-tocopherol was more effective in protecting soybean oil from oxidation than .gamma.- and .delta.-tocopherol. Jung II used a similar experimental model to assess the effects of .alpha.-, .gamma.-, and .delta.-tocopherol on chlorophyll b-photosensitized oxidation of soybean oil. They observed that as the concentrations of tocopherols increased, PV decreased and headspace oxygen increased. The .alpha.-tocopherol showed highest and .gamma.-tocopherol showed lowest antioxidant effects on photosensitized lipid oxidation. Jung II confirmed that .alpha.-tocopherol quenched singlet oxygen to reduce photosensitized lipid oxidation.
3. Measurements of Fat/Oil Oxidative Stability
Oxidative stability is defined as quantitative measurement of the susceptibility of an oil to autoxidative breakdown. The edible oil industry has long looked for a rapid analytical method to measure the oxidative stability of oils/fats. Active Oxygen Method ("AOM") is the most widely used test. The modified versions of AOM such as the Rancimat and Oil Stability Instrument methods are commonly used in the oil industry due to their convenience, even though their results are not consistent with the AOM values. Three methods based on oxygen consumption or oxygen intake are commonly employed; these methods include gravimetric measurement and measurement of oxygen consumption or pressure change in the headspace of a closed oil container. Gas chromatographic ("GC") methods determine the oxidative stability of oils by examining the concentration changes over time of certain volatile compounds in the headspace of a closed oil sampler held at an elevated temperature. A thin-film method speeds up the oxidative stability measurement by exposing an oil sample to ultraviolet ("UV") radiation; PV change in the oil over time is used to interpret the oxidative stability of oil (Gordon et al., "Assessment of Thin-Film Oxidation with Ultraviolet Irradiation for Predicting the Oxidative Stability of Edible Oils," J. Am. Oil Chem. Soc., 71:1309-1313 (1994) ("Gordon"), which is hereby incorporated by reference; Hill).
Active Oxygen Method (AOM)
AOM measures the time in hours required for a sample of fat/oil to attain a predetermined peroxide value (PV 100 meq/kg) under specific conditions. The oil/fat sample (5 g) is bubbled with dried air at a flow rate of 140 mL/min at a temperature of 98.7.degree. C. (AOCS, Official and Tentative Methods of Analysis of the American Oil Chemists Society, American Oil Chemists Society, Champaign, Ill. 1993); Laubli). The progress of oxidation is monitored by periodic examination of PV in the testing fat/oil. This method is time-consuming, labor intensive, and cost inefficient (deMan).
The Rancimat and Oil Stability Instrument
Rancimat and Oil Stability Instrument are two modified and automated versions of AOM but differ in several ways. Both methods measure the conductivity in deionized water as it increases due to the absorption of volatile acids and the decomposed products of fat/oil oxidation. Increasing conductivity is an indication of peroxide breakdown that occurs at the same time as PV increases (AOCS, Official and Tentative Methods of Analysis of the American Oil Chemists Society, American Oil Chemists Society, Champaign, Ill. 1993); Laubli).
The end-points of the automated AOM are indicated by fast increases in conductivity in the water solutions due to the rapid production of volatile fatty acids by the oils at the end of the induction period. The volatile fatty acids produced by several oils include formic, acetic, propionic, butyric, valeric, and caproic acids, among which formic acid is the major component and acetic and caproic acids are present in significant amounts. Production of formic acid is due mainly to peroxidation of aldehydes during autoxidation of oil. The increase in the formic acid concentration in the water solutions through which gas bubbled through the oil exits is responsible for the increase in their conductivity (deMan).
The Oil Stability Instrument uses a similar instrumental design as the Rancimat to measure oxidation stability of oils but is modified in many areas. The Rancimat contains only six sample holders but the Oil Stability Instrument can handle twenty-four at one time. The Rancimat has only one aluminum heating block while the Oil Stability Instrument has two, which can be set at two different temperatures. The Rancimat uses sophisticated glass joints which are replaced with rubber tubing or disposable glass by the Oil Stability Instrument. Any sample in the Oil Stability Instrument can be started and stopped at any time; this is impossible with the Rancimat. Data in the Oil Stability Instrument can be stored and handled by a personal computer, while the Rancimat is limited to its electronic recorder which stores only a set of non-transferrable data. Basically, the Oil Stability Instrument has the same application and precision as the Rancimat method (Hill).
Laubli analyzed oil stability of different oils at 100, 110, and 120.degree. C. by AOM and Rancimat methods. The results of the Rancimat method were correlated highly with those of the AOM (r=0.987) at all temperatures. The temperature coefficient of the induction time for a temperature change of 10.degree. C. ranged between 1.8 and 2.1 with a regression coefficient better than 0.99 among all the different oils.
Hasenhuettl et al., "Temperature Effects on the Determination of Oxidative Stability with the Metrohm Rancimat," J. Am. Oil Chem. Soc., 69:525-527 (1992) ("Hasenhuettl"), which is hereby incorporated by reference, observed that the logarithm of induction time of different oils analyzed by the Rancimat method was correlated highly with reaction temperature up to 140.degree. C. The conductivity curve of an oil sample tested by the Rancimat method at a reaction temperature of 150.degree. C. appeared to be inverted concave downward; this caused difficulty in placing the tangent lines. The coefficient of variation ("CV") of oxidation stability index tested by the Rancimat method was 2.3% among 6 tubes on a single test run and 10.4% among 8 different runs test at a reaction temperature of 120.degree. C. They noted that temperature variations in heating blocks can cause variations in the reported oil stability index. A collaborative study of reproducibility of the Rancimat method showed an 11.3% CV with a reaction temperature of 110.degree. C. (Hill).
Volatile antioxidants are ineffective at typical Oil Stability Instrument and Rancimat operation temperatures. Reynhout, "The Effect of Temperature on the Induction Time of a stabilized Oil," J. Am. Oil Chem. Soc., 68:983-984 (1991) ("Reynhout"), which is hereby incorporated by reference, used the Rancimat method to measure the induction time of oil treated with different types of antioxidants, including BHT, BHA, TBHQ, Herbalox (a rosemary extract), and tocopherols. Soybean oil was treated with 200 ppm of synthetic antioxidant or 400 ppm natural antioxidant. The induction time of the oil treated with BHT, BHA, and tocopherols did not appear to be different than that of the control. The oil treated with TBHQ had a longer induction time in comparison to the other treatments at different temperatures. The induction time of oil treated with Herbalox was higher than those of other treatments except for the oil treated with TBHQ. Use of the Rancimat method at 80.degree. C. to assess the effectiveness of volatile antioxidants in oil/fats improved the accuracy of the results. However, the induction times of an oil sample tested by the Rancimat at 80.degree. C. are 2 to 6 times longer than that tested at 100.degree. C. (Gordon).
Both the Rancimat and Oil Stability Instrument have limitations when analyzing as oil such as olive oil with a high AOM value for which the reported AOM value is more than 200 hr. During a long analysis procedure, the receiving water in the Rancimat or Oil Stability Instrument tends to evaporate and limits the accuracy of the instruments. Both Rancimat and Oil Stability Instrument methods use different conditions than those specified by the official AOCS method; this creates a wide variability in their reported AOM time (Hill).
Schaal Oven Test
The Schaal oven test measures the induction time both chemically (PV greatly increases) and organoleptically (the time where the first sign of rancidity occurs) in order to measure the oxidative stability of oil. In the Schaal oven test, a 100 g sample of oil or food containing oil is sealed in a bottle and placed in a dry cabinet at 65.degree. C. The sample is checked periodically by both organoleptic observation until the first sign of rancid odor is noticed and PV increases; this time is determined as the induction time (Hill).
Thin Film Ultraviolet Irradiation Method
Gordon predicted the oxidative stability of edible oils by a thin-film oxidation method accelerated by ultraviolet ("UV") irradiation. They compared the induction time of edible oils analyzed by thin-film oxidation with ultraviolet irradiation and the Rancimat methods. The GC-headspace volatiles, PVs, and conjugated dienes in the oil samples increased as the time of their exposure to the UV light increased; the induction time of the thin-film oxidation method is determined by the mean of induction time obtained by those three measurements. The mean induction time of oil samples obtained by the thin-film oxidation method were highly correlated (r=0.99) with the those tested by the Rancimat method at both 80.degree. C. and 100.degree. C., except for cocoa butter. Cocoa butter may contain more carotenes and chlorophyll than the refined oils, causing acceleration of the oil oxidation in the presence of light. They noted that the Rancimat test gives the order of stability as cocoa butter&gt;&gt;olive oil&gt;rapeseed oil&gt;corn oil&gt;soybean oil&gt;sunflower oil&gt;safflower oil. The oxidative stability of oil samples as measured by the thin film UV irradiation method was in the order of cocoa butter&gt;&gt;rapeseed oil.about.olive oil&gt;soybean oil&gt;corn oil.about.safflower.about.sunflower oil.
Thermogravimetric ("TGA") Method
Oxidation of fats/oils can be indicated by the weight change of the fats/oils in a hot air oven. TGA can be measured isothermally or dynamically under a stream of flowing hot air. Many factors, such as surface to volume ratio, temperature of the oven, air flow rate, method of sampling, and sample size affect the accuracy of the TGA method (Garcia-Mesa; Mikula). However, neither the accuracy nor reproducibility of the TGA method was reported in these articles.
Mikula analyzed the oxidative stability of soybean oils by the TGA method with a highly sensitive electronic balance. They noted that a typical thermogravimetric curve of a soybean oil sample analyzed isothermally at 150.degree. C. consisted of three phases. The first phase was the induction period ("IP"), during which only minimal weight change was observed. Rapid weight increases occurred in the second phase ("Phase II") of this analysis. Upon reaching the maximum weight gain, the weight of the testing oil began decreasing ("Phase III"). The IP, measuring the resistance of the oil to oxidation, is determined by extrapolating the baseline and upward portion of the curve until they intersect. Using the same size of platinum sample pan, IP decreased with decreased sample weight because the smaller sample exposed a greater surface area per volume than that with a larger weight. The maximum slopes of upward and downward curves were called R.sub.wg and R.sub.wl, respectively. The R.sub.wg /R.sub.wl ratio increased with sample weight. They examined the effects that temperatures between 80 and 190.degree. C. imposed on the results of the TGA test. They observed that the IP was inversely proportional to the reaction temperature. The IP had a linear relationship with the reciprocals of temperatures between 80 and 150.degree. C., giving an activation energy of 21 kcal/mole. The activation energy changed at temperatures above 150.degree. C. After they compared the results between isothermal and dynamic TGA, they concluded that the TGA obtained isothermally at 150.degree. C. was more suitable than dynamic TGA for rapid and routine evaluation of oxidative stability of freshly processed oils.
Garcia-Mesa evaluated the factors affecting the gravimetric determination of the oxidative stability of oils including reaction temperature and sample surface-to-volume ratio. They presented a different model: the weight of the oil sample increased with a corresponding increase of PV until the degradation of peroxides began. After the degradation of peroxides further weight gain of the tested oil was only slight. An increase in ether oven temperature or surface/volume ratio accelerated the oxidation process.
The precision of the gravimetric test was only legitimate up to an oven temperature of 100.degree. C.; the results of the test were not reproducible at an oven temperature greater than 100.degree. C. Limitations of this test are that discontinuous heating of the sample may affect the reproducibility of the results; the method involves intensive human work, and working conditions, including sample size, dimensions of container, and oven temperature, may cause variations in the results (Garcia-Mesa).
Oxygen Absorption Method
Drozdowski devised a rapid method to evaluate the oxidation stability of oils/fats by measuring the consumption of headspace oxygen based on the pressure difference on a manostatic device. They found that oxygen consumption in the tested oil increased as the PV of the oil increased up to the point where peroxides began degrading. Increase in the reaction vessel temperature reduced the induction time of the tested oil. The initial oil with higher PV had a shorter induction time than those with lower initial PVs. However, they analyzed a set of four low erucic acid rapeseed oils ("LEAR") with similar IVs and PVs. The results of the rapid instrumental method showed significant differences in the oxygen absorption curves among the four oils and indicated that those oils were different in oxidation stability.
Gas Chromatographic ("GC") Methods
As mentioned in the previous section, Jung II examined the oxygen consumption from the headspace of an oil sample over storage time by GC to determine the antioxidative effects of tocopherols on lipid oxidation (Jung I; Jung II). Examination of fatty acid profiles in an oil sample by GC is commonly used to determined the oxidative stability of an oil/fat due to oil formula, processing and storage conditions, or additive treatment (Cuesta; Lin; Smith). These GC methods can be rather complex and time consuming (Hill).
Snyder measured total volatiles in vegetable oils stored at 60.degree. C. for 0, 8, and 16 days, using GC-headspace analysis; the results were compared to the oxidation levels in the oil samples as indicated by PV. Total volatiles were determined by the total peak areas of volatile components on a GC chromatogram. They noted that the total volatiles for each oil tested increased with storage time at 60.degree. C. following a trend similar to PV. The production of total volatile compounds during storage of the oils was related to their fatty acid compositions. Safflower, sunflower, corn, and cottonseed oil, which have the highest amounts of linoleic acid among all oil samples, formed more volatile compounds than canola, soybean, and olive oils, which have lower levels of linoleic acid than the previously listed oils. Pentane, hexanal, and 2-pentenal, formed from oxidative decomposition of linoleic acid, showed the greatest increases during storage. The 2,4-heptadienals, oxidative decomposition products of linolenic acid and found in soybean and canola oils, increased after extended storage. VDP such as heptanal, octanal, and nonanal produced by oxidation of oleic acid did not increase between 8 and 16 days of storage.
4. Measurements of Frying Fat Deterioration
Measurements for frying fat deterioration usually are based on the physical or chemical changes of the frying oils/fats during deep-fat frying. These measurements include free fatty acid level, peroxide value, iodine value, diene concentration, refractive index, viscosity, color, Kreis test, anisidine value, levels of carbonyls, non-urea-adduct forming esters, oxirane compounds, petroleum ether-insoluble oxidized fatty acids, total polar components, and dielectric constant. The factors affecting the rate of fat deterioration during deep-fat frying are complex and yield neither a single measurement procedure leading to reliable results in all situations nor an ideal chemical method correlating well with changes in organoleptic properties of oxidized lipids throughout the entire course of autoxidation (Fritsch; Gray, "Measurement of Lipid Oxidation: A Review,"J. Am. Oil Chem. Soc., 55:539-546 (1978) ("Gray"), which is hereby incorporated by reference).
During deep-fat frying, a fat/oil is exposed to air, moisture, and high temperatures. The moisture causes hydrolysis of triglycerides and produces free fatty acids and mono- and diglycerides. The air, incorporated into the fat/oil, initiates an oxidative reaction and induces the formation of hydroperoxides, conjugated dienic acids, epoxides, hydroxides, and ketones on one or more of the fatty acid chains of the triglyceride. The oxidized triglyceride may continue breaking down into smaller fragments or may undergo polymerization with other oxidation products into dimeric and higher polymeric triglycerides. High frying and cooking temperatures (170-200.degree. C.) also trigger the thermal decomposition and polymerization in the fats/oils (Arroyo; Boskou; White, "Methods for Measuring Changes in Deep-Fat Frying Oils," Food Technol., 45:75-80 (1991), which is hereby incorporated by reference). these reactions cause changes of the functional, sensory, and nutritional properties in frying fats and may lead to a point where the quality of fried foods is no longer acceptable and where frying fats should be discarded.
As mentioned in the previous section, decomposition compounds of oil/fat oxidation are categorized into VDP and NVDP; these compounds usually are used as the indicators of oil/fat deterioration. Chang et al., "Chemical Reactions Involved in the Deep-Fat of Frying Foods," J. Am. Oil Chem. Soc., 55:718-727 (1978), which is hereby incorporated by reference, measured the VDP collected from a simulated deep-fat frying system using corn oil, hydrogenated cottonseed oil, trilinolein, and triolein by a GC method. A total of 220 compounds were identified. Because measurement of VDP is very time-consuming and complicated, little additional work has been conducted with VDP since the 1970s. The formation and accumulation of NVDP change the physical and chemical properties of frying fat/oil. Physical changes include increases in viscosity, color, dielectric constant, and foaming, and a decrease in smoke point. Chemical changes involve increases in FFA, PV, conjugated dienoic acids, TPC, polymers, carbonyl value, hydroxyl content, saponification value, and a decrease in unsaturation (Fritsch; White). Many factors such as type of fat, type of food, conditions of operation and dryer can cause variations in the physical and chemical changes occurring in frying fat during deep-fat frying; the variations make it difficult to select any standard to interpret the results across different types of oils and frying applications (Fritsch). However, no single test can be used universally to determine the point where a frying fat needs to be discarded (Melton et al., "Review of Stability Measurements for Frying Oils and Fried Food Flavor," J. Am. Oil Chem. Soc., 71:1301-1308 (1994) ("Melton"), which is hereby incorporated by reference).
Cut-off levels are the maximum and minimum acceptable values for the fat to be considered as good quality. Examples include a 1% level of petroleum ether-insoluble oxidized fatty acids or a smoke point of 170.degree. C. Alternatively, a TPC level of 27%, corresponding to 1% level of petroleum ether-insoluble oxidized fatty acids, also is commonly accepted by the edible oil industry as a critical indicator for the quality control of frying oil (Fritsch).
Peroxide Value
Hydroperoxides are the primary products of lipid oxidation and generally are referred to as peroxides. The concentration of peroxides may be used as an assessment of the degree of lipid oxidation. However, hydroperoxides are very unstable and sensitive to temperature changes; they readily decompose into carbonyl and hydroxyl compounds. PV of an oil sample continues to increase after the sample is removed from the fryer. The PV method is limited in that it measures only the initial stage of oxidation; the PV increases initially then decreases over the entire course of oxidation (Gray).
Thiobarbituric Acid ("TBA") Test
The TBA method is based on the color formation between two TBA molecules and one molecule of malonaldehyde. Malonaldehyde is a secondary product of oxidation of polyunsaturated fatty acids with two or more double bonds. Formation of malonaldehyde, is achieved by cyclization of the peroxide group and the .beta. and .gamma. double bonds next to the peroxide group. Oxidized lipid lacking polyunsaturated fatty acids such as linoleate forms no color reaction with TBA even at a PV of 2000 or greater (Gray).
Non-volatile Carbonyl Compounds
Carbonyl compounds, which are degraded compounds from hydroperoxides, are secondary products of lipid oxidation. Non-volatile carbonyl compounds are probable flavor precursors to more volatile compounds, but they yield no direct contribution to the flavor. The most reliable method for measuring non-volatile carbonyl compounds is based on the reaction of saturated and unsaturated aldehydes with anisidine (p-methoxyaniline). A high correlation between the anisidine values of salad oils and their flavor scores has been reported (Gray). A multiple correlation showed a correlation coefficient of 0.81 between flavor scores, anisidine values, and peroxide values; however, this method, as the peroxide value, is limited to measuring the early stage of lipid oxidation.
Oxirane Determination
Oxirane compounds, containing .alpha.-epoxy groups, are formed during oxidation of unsaturated lipid material. The (.alpha.-epoxy groups are measured by the consumption of a halogen by a fat sample reacting with a known excess amount of halogen in a suitable solvent. This method is reported to be particularly suitable for determination of epoxides in heated fats in which the oxirane level is less than 0.1% (Gray).
Conjugated Diene Method
Oxidation of polyunsaturated fatty acids causes formation of conjugated unsaturated fatty acids which exhibit strong absorption from 230 to 375 nm. Conjugated diene and triene unsaturation have maximum absorptions at 234 nm and 268 nm, respectively. Increase in the absorption at 234 nm usually is used to indicate the degree of oxidation of a fat sample containing linoleate or a more highly unsaturated fatty acid. The magnitude of the UV absorption is not related to the degree of oxidation because different fatty acids vary in their absorption at 234 nm. However, the change of absorption at 234 nm of a given fat sample can be used as a relative measure of oxidation. This test is most useful in measuring heat abuse of polyunsaturated oils, but is less applicable to fat containing few unsaturates (Gray; Peled et al., "Effect of Water and BHT on Stability of Cottonseed Oil During Frying," J. Sci. Food Agric., 26:1655-1659 (1975), which is hereby incorporated by reference).
Refractometry
Refractive indices of fats/oils increase upon autoxidation. The change in refractive index of a fat sample follows the three stages of fat/oil oxidation. During the induction period, the peroxide formation is low and the refractive index remains constant. In the secondary stage, the refractive index increases sharply as the peroxide value increases before reaching the maximum point. The development of conjugated unsaturation contributes to the increase in refractive index during this stage of oxidation. In the tertiary stage, where the peroxides decompose, the refractive index continues to increase at a steady rate, which is less than that in the secondary stage. Polymerization of partially oxidized fats is responsible for this increase in refractive index during the tertiary stage of oxidation (Arya et al., "Refractive Index as an Objective Method for Evaluation of Rancidity in Edible Oils and Fats," J. Am. Oil Chem. Soc., 46:28-30 (1969), which is hereby incorporated by reference).
Total Polar Components
Determination of TPC has proved to be accurate, simple, and reproducible (Dobarganes et al., "High Performance Size Exclusion Chromatography of Polar Compounds in Heated and Non-Heated Fats," Fat Sci. Technol., 90:308-311 (1988), which is hereby incorporated by reference). Total polar materials are determined by dissolving a weighed amount of fat (2.5 g) in light petroleum ether:diethyl ether (87:13) and passing it through a silica gel column that absorbs the polar compounds. After evaporation of the eluted solvent, the nonpolar fat is weighted and the total polar material is estimated by difference. A level of 27% TPC has been suggested as the upper limit to discard a frying fat (Paradis et al., "Evaluation of New Methods for Assessment of Used Frying Oils," J. Food Sci., 46:449-451 (1981); Paradis et al., "A Gas Chromatography Method for the Assessment of Used Frying Oils: Comparison With Other Methods," J. Am. Oil Chem. Soc., 58:635-638 (1981) ("Paradis"), which are hereby incorporated by reference).
Fatty Acid Analysis and 18:2/16:0 Ratio
Thompson et al., "Lipid Changes in French Fries and heated Oils During Commercial Deep Frying and Their Nutritional and Toxicological Implications," Can. Inst. Food Sci. Technol. J., 16:246-253 (1983), which is hereby incorporated by reference, found that a lightly hydrogenated frying oil, after 100 hr of frying, had 50% less total linoienic and linoleic acids than the fresh oil. Thus, the relative amount of saturated fatty acids increased. Miller et al., "High-Temperature Stabilities of Low-Linolenate, High-Stearate and Common Soybean Oils," J. Am. Oil Chem. Soc., 65:1324-1327 (1988), which is hereby incorporated by reference, also reported a decrease in linoleic and linolenic fatty acids and an increase in relative amounts of saturated fatty acids in soybean oils heated at 180.degree. C. for 40 hr.
Gas Chromatographic Method
Paradis evaluated the quality of used frying oil by measuring the dimeric polymer content in the oil sample with a GC. Corn oil was heated at 185.degree. C. for various periods of time followed by chemical analysis. Percentage of dimeric polymers in the total triglycerides, total polar compounds, and dielectric constant of the corn oil increased over the heating time but no correlation among those three methods was tested. The GC method had the highest correlation coefficient between the values and heating times among the three methods.
High Performance Size-exclusion Chromatographic Method ("HPSEC")
White et al., "A High Performance Size-Exclusion Chromatographic method for Evaluating Heated Oils," J. Am. Oil Chem. Soc., 63:914-920 (1986) ("White"), which is hereby incorporated by reference, measured the changes of polymer content in heated oils by the HPSEC method to determine the quality of frying oils. Four peaks were found in the heated soybean oils by HPSEC. Peaks 1, 2, 3, and 4 were referred to as triglycerides ("TG") and fatty acids, dimeric TG, tetrameric TG, and polymers larger than tetrameric TG, respectively. Only peaks 1 and 2 were present in unheated oils. Areas of peaks 1, 2, and 3 behaved less predictably than that of peak 4. The area of peak 4 increased continuously over entire heating periods while the areas of peaks 1, 2, and 3 tended to increase in the early stage of heating then decrease toward the end of the heating period. The increases in the areas of peak 4 over heating time were highly correlated with the total polar compounds in the heated oils; a 27% level of total polar compounds, the recommended level for discarding a frying fat, corresponded to a 5 cm.sup.2 area of peak 4 when a 10 .mu.g sample was injected. The authors also provided the following experiment to demonstrate the relationship between the HPSEC method and the total polar compounds in the heated oils. The non-polar fraction ("F-1") and polar fraction ("F-2") of a heated oil sample eluted by the column chromatographic method were run by HPSEC; the F-1 of a heated oil contained only peak 1, while all four peaks occurred in the F-2. The peak 1 area of F-1 of the heated oil decreased gradually through the entire heating time, but peak 1 area of F-2 increased in the early stage of the heating period then leveled off toward the end of heating period. The authors reported also that most conjugated TG in the fresh oils were weakly oxidized and were thus non-polar. Decrease of unoxidized TG (peak 1) from F-1 was accompanied by an increase of oxidized TG that appeared as peak 1 in the F-2. areas of all peaks in F-2 increased then leveled off during heating; this was caused by incomplete recovery of polar compounds after 42 hr of heating. The magnitude of increase in peak 4 area in F-2 of a heated oil was much greater than that of other peaks; this corresponds to the increase in the total polar compounds in the heated oil. One of the advantages of this method is its speed; it requires 2 min of sample preparation and 20 min of HPSEC run time.
Petroleum Ether-Insoluble Oxidized Fatty Acids Method
It is recommended that a frying fat be discarded if the concentration of petroleum ether-insoluble oxidized fatty acids ("PEIOFA") is 1.0% higher. The PEIOFA is determined by the following steps. A frying fat sample is saponified with potassium hydroxide in ethanol, and the soap solution is acidified and thoroughly extracted with ether. After evaporation of solvent, the mixture of fatty acids is reextracted with boiling petroleum ether. The ratio of weight of remaining petroleum ether insoluble material to the sample weight is determined as PEIOFA. Billek et al., "Quality Assessment of Used Frying Fats: A Comparison of Four Methods," J. Am. Oil Chem. Soc., 55:728-733 (1978), which is hereby incorporated by reference, compared the results of frying fat quality measured by the PEIOFA method with those measured by gel permeation chromatography ("GPC"), liquid chromatography ("LC"), and column chromatography ("CC") methods. They concluded that 1% of PEIOFA in frying fat corresponded to 15% of polymeric triglycerides measured by the GPC method, 28% of total polar artifacts measured by LC method, and 27% of total polar components measured by CC methods, respectively.
Gray reviewed measurements of lipid oxidation. He noted that no single chemical method correlated with changes in organoleptic properties of oxidized lipids throughout the entire course of autoxidation. As mentioned earlier, Smith surveyed the qualities of used frying fat from fast food restaurants by evaluating the dielectric constant, TPC, FFA, and fatty acid profiles. In general, they found close correlations between frying times and increases in dielectric constant (r=0.84), percentage of TPC (r=0.80), and percentage of FFA (r=0.88). The correlation coefficients between increase in dielectric constant and TPC, and between increase in dielectric constant and FFA were 0.93 and 0.92, respectively, and showed that the dielectric constant method was an easy and convenient alternative method of quality control of frying fats for a fast food restaurant. The greatest changes in fatty acid profiles occurred with trans-C18 monoenes (elaidic acid), which decreased with hours of use (r=-0.69). Palmitic acid increased (r=0.69), but stearic acid decreased (r=-0.66) over the use periods of the frying fats (Gray; Smith).
In spite of the many methods for measuring oil stability to oxidation and of many measurements of oil degradation products formed during frying, there is still a need for a rapid method for measuring stability of an oil to frying. The current methods of Oil Stability Instrument or Rancimat for measuring resistance of fats/oils to thermal oxidation still require rather long periods of time (hr) and fail to take into consideration the effect of hydrolysis on frying oil stability. These tests also do not measure the stabilizing effect of surface active agents such as siloxanes on the frying oils (Melton). therefore, a need still exists for a more rapid and reliable method for measurements of the stability of frying fats/oils and the effects of the different additives, such as antioxidants and surface active agents, on that stability.